A single retroviral protein species, the Gag polyprotein, is sufficient for assembly of retrovirus particles. Since this process includes the selective encapsidation of viral RNA, this protein is evidently capable of specific interactions with nucleic acids. The nature of these interactions is not well understood as yet. After the virion is released from the cell, the polyprotein is cleaved by the virus-coded protease; one of the cleavage products, termed the nucleocapsid (NC) protein, then binds to the genomic RNA, forming the ribonucleoprotein core of the mature particle.
The interaction between Gag and genomic RNA is known to involve the NC domain of the polyprotein, since mutants within NC are defective in RNA packaging and since the specificity of encapsulation tends to be determined by the NC domain in chimeric Gag molecules. However, NC is a basic protein and has frequently been described as binding to single-stranded DNA or RNA in a sequence-independent manner. Indeed, it has been hypothesized to be capable of binding to any single-stranded nucleic acid under appropriate conditions. This binding activity appears to be important at several stages of virus replication.
A search of all known retroviruses reveals a highly conserved structure in their NC proteins. All NC proteins of the Oncovirinae and Lentivirinae subfamilies of Retroviridae contain one or two copies of a conserved sequence motif termed the "cysteine array" or "Cys-His box." This motif can be represented as Cys(X).sub.2 Cys(X).sub.4 His(X).sub.4 Cys (SEQ ID NO:1) (Henderson et al., J. Biol. Chem., 256:8400 (1981)). This motif is also known as the NC zinc finger or, alternatively, as the retroviral CCHC (SEQ ID NO:2) zinc finger because it chelates zinc through histidine imidazole and cysteine thiolates with a K.sub.d less than 10.sup.-13 (Berg, Science, 232:485 (1986); Bess, Jr., et al., J. Virol., 66:840 (1992); Chance, et al., Proc. Natl. Acad. Sci. U.S.A., 89:10041 (1992); South, et al., Adv. Inorg. Biochem., 8:199 (1990); South, et al., Biochem. Pharmacol., 40:123 (1990)). Examples of retroviruses which possess at least one CCHC (SEQ ID NO:2) type zinc finger per nucleocapsid protein include, but are not limited to, HIV-1, HIV-2, SIV, BIV, EIAV, Visna, CaEV, HTLV-1, BLV, MPMV, MMTV, RSV, MuLV, FeLV, BaEV, and SSV.
The function of the NC zinc finger is not yet fully understood. However, all mutations in the zinc-binding residues which been described to date have been lethal for the virus. Virions produced by these mutants are frequently defective with respect to genomic RNA content (Aldovini, et al., J. Virol., 64:1920-1926 (1990); Dorfman, et al., J. Virol., 67:6159-6169 (1993); Dupraz, et al., J. Virol., 64:4978-4987 (1990); Gorelick, et al., Proc. Natl. Acad. Sci. USA, 85:8420-8424 (1988); Gorelick, et al., J. Virol., 46:3207-3211 (1990); Meric, et al., J. Virol., 63:1558-1568 (1989). As such, it is thought that the zinc fingers participate (as part of the Gag polypeptide precursor) in RNA packaging during virion assembly. Significantly, however, the mutant particles are far more defective with respect to infectivity than with respect to genomic RNA content (Gorelick, et al., Proc. Natl. Acad. Sci. USA, 85:8420-8424 (1988)). A new class of zinc-finger mutants of Moloney MuLV (Mo-MuLV) have recently been characterized (Gorelick, et al. (1996) J. Virol. 70:2593-2597), and indeed, it has been found that these mutants package normal levels of genomic RNA, but are nevertheless noninfectious. Such observations imply that the zinc fingers play other roles in the viral life cycle in addition to their function in RNA packaging. It has been suggested that NC has an important role in maturation of the released virus particle (Fu, et al., J. Virol., 68:5013-5018 (1994); Fu and Rein, J. Virol., 67:5443-5449 (1993)), and performs one or more functions in reverse transcription (Allain, et al., EMBO J., 13:973-981 (1994); Lapadat-Tapolsky, et al., Nucleic Acids Res., 21:831-839 (1993); Nagy, et al., J. Virol., 68:757-765 (1994); Peliska, et al., Biochemistry, 33:13817-13823 (1994); Roberts, et al., Biochem. Biophys. Res. Commun., 160:486-494 (1989); Rodriguez-Rodriguez, et al., J. Biol. Chem., 270:15005-15011 (1995); You, et al., J. Biol. Chem., 269:31491-31495 (1994); Wu et al. (1996) J. Virol. 7132-7142). To date, however, the significance of the zinc finger and, in turn, the NC protein is not fully understood, nor is the binding of NC to nucleic acids.